U.S. patent application number 12/531001 was filed with the patent office on 2010-07-22 for improving cold- and salt-tolerant performance of plants with transcription factor gene snac2 from rice.
This patent application is currently assigned to Huazhong Agricultural University. Invention is credited to Honghong Hu, Lizhong Xiong.
Application Number | 20100186108 12/531001 |
Document ID | / |
Family ID | 38770851 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100186108 |
Kind Code |
A1 |
Hu; Honghong ; et
al. |
July 22, 2010 |
Improving Cold- and Salt-tolerant Performance of Plants with
Transcription Factor Gene SNAC2 from Rice
Abstract
The present invention relates to clone isolation, function
confirmation and use of the SNAC2 gene from rice associated with
the plant tolerance to cold and salt stress. Said gene comprises
(a) a DNA sequence as shown in position 112-1023 of SEQ ID NO:1, or
(b) a DNA sequence that encodes the same protein as that encoded by
(a). The present invention also relates to use of said gene in
increasing the tolerance of plants to drought and salt stress.
Inventors: |
Hu; Honghong; (Wuhan,
CN) ; Xiong; Lizhong; (Wuhan, CN) |
Correspondence
Address: |
THOMPSON COBURN, LLP
ONE US BANK PLAZA
ST. LOUIS
MO
63101
US
|
Assignee: |
Huazhong Agricultural
University
|
Family ID: |
38770851 |
Appl. No.: |
12/531001 |
Filed: |
March 11, 2008 |
PCT Filed: |
March 11, 2008 |
PCT NO: |
PCT/CN08/00483 |
371 Date: |
February 19, 2010 |
Current U.S.
Class: |
800/278 ;
800/298 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8273 20130101; C07K 14/4702 20130101 |
Class at
Publication: |
800/278 ;
800/298 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/82 20060101 C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2007 |
CN |
200710051654.7 |
Claims
1. A transformed plant comprising a recombinant DNA construct
comprising a promoter functional in a plant cell positioned to
provide for expression of a polynucleotide having a sequence with
at least about 70%, 75%, 80%, 85%, 90%, or 95% identity to those in
positions 112-1023 of SEQ ID NO:1.
2. A transformed plant comprising a recombinant DNA construct
comprising a promoter functional in a plant cell positioned to
provide for expression of a polynucleotide encoding a polypeptide
at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to that
encoded by the polynucleotide sequence in positions 112-1023 of SEQ
ID NO:1.
3. The transformed plant according to claim 1, wherein said plant
is a crop plant.
4. A method of producing a transformed plant having an improved
property, wherein said method comprises transforming a plant with a
recombinant construct comprising a promoter functional in a plant
cell positioned to provide for expression of a polynucleotide
encoding a polypeptide useful for improving plant cold tolerance,
drought tolerance, or salt tolerance, wherein said polynucleotide
has a sequence with at least about 70%, 75%, 80%, 85%, 90%, or 95%
identity to those in positions 112-1023 of SEQ ID NO:1.
5. A method of producing a transformed plant having an improved
property, wherein said method comprises transforming a plant with a
recombinant construct comprising a promoter functional in a plant
cell positioned to provide for expression of a polynucleotide
encoding a polypeptide useful for improving plant cold tolerance,
drought tolerance, or salt tolerance, wherein said polypeptide is
at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to that
encoded by the polynucleotide sequence in positions 112-1023 of SEQ
ID NO:1.
6. The method according to claim 4, wherein said transformed plant
is a crop plant.
7. A plant exhibiting an improved property as compared to the
control plant, wherein the altered trait is selected from the group
consisting of greater cold tolerance, greater tolerance to water
deprivation, and greater salt tolerance, or combinations thereof,
wherein the plant has greater expression or activity of a
polypeptide encoded by a polynucleotide that has at least about
70%, 75%, 80%, 85%, 90%, or 95% identity to those in positions
112-1023 of SEQ ID NO:1.
8. A plant exhibiting an improved property as compared to the
control plant, wherein the altered trait is selected from the group
consisting of greater cold tolerance, greater tolerance to water
deprivation, and greater salt tolerance, or combinations thereof,
wherein the plant has greater expression or activity of a
polypeptide at least about 70%, 75%, 80%, 85%, 90%, or 95%
identical to that encoded by the polynucleotide sequence in
positions 112-1023 of SEQ ID NO:1.
9. The transformed plant according to claim 7, wherein said plant
is a crop plant.
10. The transformed plant according to claim 1, wherein said plant
has enhanced salt resistance.
11. The transformed plant according to claim 1, wherein said plant
has enhanced cold resistance.
12. The transformed plant according to claim 1, wherein said plant
has enhanced salt resistance and enhanced cold resistance.
13. The transformed plant according to claim 1, wherein said
polynucleotide comprises a sequence of positions 112-1023 of SEQ ID
NO:1.
14. The transformed plant according to claim 2, wherein said plant
has enhanced salt resistance.
15. The transformed plant according to claim 2, wherein said plant
has enhanced cold resistance.
16. The transformed plant according to claim 2, wherein said plant
has enhanced salt resistance and enhanced cold resistance.
17. The transformed plant according to claim 2, wherein said
polynucleotide encodes a polypeptide identical to that encoded by
the polynucleotide sequence in positions 112-1023 of SEQ ID
NO:1.
18. The transformed plant according to claim 2, wherein said plant
is a crop plant.
19. The method according to claim 5, wherein said transformed plant
is a crop plant.
20. The transformed plant according to claim 8, wherein said plant
is a crop plant.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of plant
biotechnology, and more particularly, to clone isolation, function
confirmation and use of a DNA fragment (gene) from rice. Said gene
is associated with plant tolerance to cold and salt stress. The
cold- and salt-tolerance performance of transgenic plant is
markedly improved by transferring a complete translation region
(Coding sequence) of the gene linked with a strong promoter
(Ubiquitin1) from corn to a plant.
BACKGROUND ART
[0002] The growth of plants is naturally subjected to the influence
of environment. For example, drought, salt injury and low
temperature always lead to great reduction of crop production,
thereby posing a challenge to the development of agriculture in
many areas. To cope with or adapt to the adverse effect of
environment, plants perceive the changes of extracellular
environmental conditions and signal the cells through many
pathways. In response, the cells induce expression of some
responding genes to generate some functional proteins,
osmoregulation substances and transcription factors for signal
transmission and gene expression regulation, all of which protect
the cells from stress impairment of drought, high salinity, low
temperature and the like, so that plants are able to make
corresponding responses to environmental changes (Xiong et al.,
Cell signaling during cold, drought and salt stress. Plant Cell. 14
(suppl), S165-S183, 2002). Whether those functional genes can be
properly expressed during a response to an environmental stimulus
is precisely regulated by regulation factors, particularly,
transcription factors. It has been currently found that expression
of members of transcription factor families such as AP2/EREBP, Zinc
finger, Myb, bZIP, and NAC may be induced or inhibited under
different environmental stresses. Therefore, they are considered to
play a very important role in regulating and controlling a plant's
response to environmental stress. Moreover, the isolation and
identification of the transcription factors, which play a critical
role in regulation and control and may be applied to genetically
improve stress-resistant crops, is highly contributive to crop
breeding. Presently, attempts have been made to improving plant
performance during stress. For example, transgenic Arabidopsis
plants overexpressing DREB1A showed increased tolerance to low
temperature and drought than wild type plants (Liu Q et al., "Two
transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA
domains separate two cellular signal transduction pathways in
drought- and low-temperature-responsive gene expression,
respectively, in Arabidopsis." Plant Cell. 1998, 10: 1391-1406).
The research group of Thomashow at Michigan State University
(U.S.A) also cultivated plants with enhanced cold tolerance via
genetic transformation with Arabidopsis CBF1 gene.
[0003] Rice is one of the most important alimentary crops. Rice
with improved cold- and salt-tolerant performance has important
significance for human. Therefore, there exists an urgent need to
find transcription factors associated with cold- and salt-tolerant
performance so as to cultivate enhanced cold- and chilling-tolerant
varieties.
SUMMARY OF THE INVENTION
[0004] An object of the present invention is to isolate a DNA
fragment containing complete encoding segments of a transcription
factor gene associated with cold and salt tolerance from rice, to
clone it, and to use the gene to improve the stress tolerance of
rice and other plants. A structure analysis of this gene has shown
that it belongs to NAC transcription factor family specific to
plant, and relates to stress, as such named SNAC2.
[0005] The present invention relates to isolation and use of a DNA
fragment containing SNAC2 gene, which confers plant enhanced
tolerance ability in stress conditions such as low temperature and
the like. Said DNA fragment is, for example, shown in SEQ ID NO: 1,
the highly homologous DNA sequence substantially equivalent to SEQ
ID NO: 1, or the subfragment of sequence shown in SEQ ID NO: 1
having substantially the same function.
[0006] A gene of the present invention or a homologous gene thereof
can be obtained by screening a cDNA or genomic DNA library with a
cloned SNAC2 gene used as a probe. The SNAC2 gene of the present
invention and any DNA segments of interest or homologous DNA
segments thereof may also be obtained by amplification from genomic
DNA, mRNA and cDNA using PCR (polymerase chain reaction)
technology. Thereby, a sequence containing SNAC2 gene may be
isolated. By transforming plants with said isolated sequence in any
expression vector that can direct the expression of an exogenous
gene in plant, transgenic plants with enhanced tolerance to low
temperature and high salinity stresses can be produced. According
to the present invention, in the process of constructing the gene
of the present application into the expression vector of the plant,
any strong promoter or inducible promoter can be added to the
position preceding the transcription initiation nucleotide, or
alternatively, an enhancer may be used. Such a enhancer region can
be ATG start code and start code of contiguous regions and the
like, provided that the enhancer region is in the same frame as the
coding sequence to ensure the translation of a complete
sequence.
[0007] The expression vector bearing a SNAC2 gene of the present
invention can be introduced into plant cells by conventional
biotechnological techniques such as Ti plasmid, plant virus vector,
direct DNA transformation, microinjection, electroporation, and the
like (Weissbach, 1998, Method for Plant Molecular Biology VIII,
Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998,
Plant Molecular Biology (2.sup.nd Edition)).
[0008] The expression vector containing a SNAC2 gene of the present
invention can be used to transform a host, which is a wide variety
of plants including rice, to cultivate plant varieties having
excellent salt- and cold-resistance.
[0009] The gene of the present invention is expressed by induction
of stress, and therefore its promoter is an inducible-type
promoter. By inserting both a promoter segment of the present
invention and any gene of interest into an appropriate expression
vector and transforming a plant host, it is feasible to induce
expression of the gene under stress conditions, thereby improving
the tolerance performance of the plants in response to stress.
[0010] The present invention will be further demonstrated hereunder
in conjunction with specific embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] SEQ ID No: 1 in the Sequence Listing shows the DNA fragment
sequence isolated and cloned in accordance with the present
invention, comprising SNAC2 gene encoding region.
[0012] FIG. 1 shows a flow chart of isolation and identification of
SNAC2 gene.
[0013] FIG. 2 shows the expression level of SNAC2 gene measured by
Northern hybridization at different time points under different
stresses including drought, salt, cold and ABA.
[0014] FIG. 3 shows the expression of SNAC2 gene in transgenic
plants, wherein with the first lane is control, and the rest are
transgenically independent transgenic plants.
[0015] FIG. 4 shows growth of seedling stage SNAC2 over-expressing
transgenic families in recovery phase after subjecting to low
temperature stress, wherein a half of each little red pail is
planted with the controls, the other half is planted with
transgenic plants. Low temperature stress refers to a condition
where the plant is subjected to 12 h light/12 h dark in a 4.degree.
C. incubator for 5 days, followed by growth recovery under normal
conditions.
[0016] FIG. 5 shows growth of seedling stage SNAC2 over-expressing
transgenic families in high salinity, wherein FIG. 5A is a picture
taken from seedlings that had been germinated for 4 days before
being transplanted to a MS medium containing 150 mM NaCl where it
grew for 18 days, and FIG. 5B shows statistical results concerning
plant height and root length (B).
[0017] FIG. 6 illustrates a trans-activation assay in yeast and a
yeast single-hybrid assay that demonstrate that SNAC2 has
characteristics of transcription activation and DNA binding,
wherein FIG. 6A is the trans-activation assay; FIG. 6 B is the
yeast single-hybrid assay.
[0018] FIG. 7 illustrates a subcellular location of SNAC2 gene in
plant cells. FIG. 7A is schematic diagram of a constructed vector;
FIG. 7B is visual inspections with confocal microscopy, among which
FIG. 7B(i) is an observation of callus section stained with
fluorescent dye propidium iodide, FIG. 7B(ii) is an image of GFP
expression under green fluorescence, FIG. 7B(iii) is merged result
of red and green fluorescences.
EXAMPLES
[0019] At the initial stage of the present invention the cDNA clone
99C10 derived from the rice variety MingHui 63 (a rice variety
which is widely cultivated in China) was obtained. This cDNA as
obtained is a full-length cDNA of SNAC2 gene, which is a
transcription factor associated with drought resistance. These are
the following reasons why this cDNA was used: (1) it was observed
upon an analysis using a cDNA chip technique that the expression
amount of cDNA clone 99C10 in the rice variety "ZhongHan 5" (a
public available rice variety provided by Shanghai Academy of
Agricultural Sciences, China) was increased by 3.5 times after a
drought stress treatment for 15 days. The results of sequencing
indicated that the gene was OsNAC6 (accession number AK068392). In
view of significant difference in the expression amount of the
clone after drought treatment and its functional characteristics,
it was considered that the gene represented by 99C10 clone was
involved in regulating and controlling expression of the gene under
stresses; (2) according to the analysis of expression profile of
the gene under stress (see FIG. 2), it was found that the
expression level of the gene was notably increase during stress
treatments; (3) the transgenic plants with over-expression of the
full length gene have significantly enhanced tolerance to cold and
high salinity (see FIGS. 4 and 5). All of these show that SNAC2
gene is a stress-associated gene involved in regulating and
controlling the resistance not only to drought but also to high
salinity and cold.
[0020] The present invention will become more apparent with the
following descriptions of the examples, which are related to the
methods for isolating and cloning the DNA fragment comprising the
complete encoding region of SNAC2 gene and for verifying the
function of SNAC2 gene based on the above results at the initial
stage of the present invention (flow scheme of these methods as
shown in FIG. 1). From the following description and examples, one
skilled in the art can determine the basic features of the present
invention, and appreciate that any changes and modifications can be
furthermore made to the present invention to adapt to various uses
and conditions without departing from the spirit and scope of the
present invention.
Example 1
Isolation and Clone of DNA Fragment Containing SNAC2 Gene
Segment
[0021] An analysis on the expression profile of drought inducible
genes of the rice variety "ZhongHan 5" (a public available rice
variety provided by Shanghai Academy of Agricultural Sciences,
China) found a strongly drought-inducible EST (expression sequence
tag), the expression amount of which increased 3.5 times or even
more in the later stage of drought stress. A sequence analysis
indicated that this gene was a member of the transcription factor
family NAC and was a full-length sequence, corresponding to cDNA
clone J013149P14 of Japan Rice full-length Database
(http://cdna01.dna.affrc.go.jp). According to this cloned sequence,
the primer T050F (5'-CAGGTACCGCCAAGCCCTCCTCTCCTCTTCCCAT-3',
sequence specific primer plus linker KpnI site) and T050R
(5'-CAGGATCCCCTCGTCGTCGTTCAGTCC-3', sequence specific primer plus
linker BamHI) were designed, and the 1-1269 by of the clone was
amplified from the variety "ZhongHan 5" by reverse transcription
(FIG. 3). The amplified product is the sequence of 1-1269 by of the
present invention. The acquisition of this full-length gene
comprised the following steps: extracting with a TRIZOL reagent
(purchased from Invitrogen Inc.) the total DNA from the leaves from
the rice variety "ZhongHan 5" subjected to drought stress treatment
(see the manual of above described TRIZOL reagent for the detailed
extraction method); reverse transcribing with a reverse
transcriptase (purchased from Invitrogen Inc.) to synthesize cDNA
first chain (reacted under 65.degree. C. for 5 min, 42.degree. C.
for 50 min, and 70.degree. C. for 10 min); amplifying the reverse
transcribed product with nested primer designed according to the
sequence of cDNA clone J013149P14 (the reaction conditions were:
predenaturation at 94.degree. C. for 2 min; 30 cycles of 94.degree.
C. for 30 sec, 55.degree. C. for 30 sec, 72.degree. C. for 2 min;
extension at 72.degree. C. for 5 min); inserting the PCR products
obtained from amplification into a pGEM-T vector (purchased from
Promega Inc.); and selecting and sequencing the positive clones to
obtain the desired full-length gene. Such a clone was named as
PGEM-SNAC2.
Example 2
Detection of Inducible Expression of Rice Endogenous Gene SNAC2
[0022] The rice variety "Zhonghan 5" as a raw material was treated
separately under drought, cold and high-salinity stress as well as
ABA at the 3 leaf stage. The drought treatment was conducted by
immersing the seedling root into 20% polyethylene glycol
(trademarked as PEG6000) for 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, before
sampling. The cold treatment was conducted by placing the seedling
in an incubator at 4.degree. C. for 0 h, 1 h, 8 h, 12 h, before
sampling. The high-salinity stress was conducted by immersing the
seedling root in 200 mM/L NaCl solution for 0 h, 4 h, 8 h, 16 h,
before sampling. The ABA treatment was conducted by immersing the
seedling root in 100 .mu.M/L ABA solution for 0 h, 0.5 h, 3 h, 6 h,
12 h, 24 h before sampling. The total RNAs of the leaves were
extracted (using Trizol reagent, Invitrogen), then subjected to RNA
membrane transfer (according to the experimental methods of
"Molecular Cloning", Science Press, Peking, 1999), and finally a
Northern hydride with SNAC2 used as a probe. The result showed that
the expression of the SNAC2 gene cloned in the present invention
can be induced by drought, cold, high-salinity and ABA (shown as in
FIG. 2), therefore considered a stress-associated transcription
factor.
Example 3
Construction and Transformation of SNAC2 Gene Over-Expression
Vector
[0023] The above Example 2 showed that the expression of the SNAC2
gene of the present invention can be induced by drought, cold,
high-salinity and ABA. In order to better illustrate the function
of this gene, the SNAC2 gene was over-expressed in rice and
verified by the phenotype of transgenic plants. The process
comprises the following steps of: double digesting the positive
clone pGEM-SNAC2 plasmid obtained in Example 1 with BamHI and KpnI;
recovering exogenous fragments; at the same time, enzymatically
cleaving the genetic transformation vector pU1301 with the corn
strong promoter Ubiquitin1 (which is reconstructed based on a
common vegetable genetic transformation vector pCAMBIA1301 from
Australia CAMBIA Laboratory (Center for the Application of
Molecular Biology to International Agriculture), carrying corn
strong promoter Ubiquitin1 with constitutive and over-expression
characteristics, mediated by Agrobacterium) by the same way;
exacting and purifying the enzymatically cleaved products with
chloroform:isopentanol (24:1) after enzymatically cleavage. The
linkage reaction was conducted by using the enzymatic cleavage
fragments comprising SNAC2 gene and the enzymatically cleaved
pU1301 vector (shown as the figure below), transforming E. coli
DH10.beta. (purchased from Invitrogen Inc.). The positive clones
were screened out by enzymatically cleavage to obtain a transformed
vector.
##STR00001##
[0024] The transformed vector was introduced into the rice variety
"ZhongHua 11" (a publicly available rice variety provided by China
National Rice Research Institute) using a rice genetic
transformation system mediated by Agrobacterium. A transgenic plant
was then finally obtained by precultivation, infestation,
co-cultivation, screening the callus with hygromycin resistance,
differentiation, rooting, seedling establishment and transplanting.
The rice (japonica rice subspecies) genetic transformation system
mediated by Agrobacterium was optimized on the basis of the method
reported by Hiei, et al. (See: Efficient transformation of rice,
Oryza sativa L., mediated by Agrobacterium and sequence analysis of
the boundaries of the T-DNA, 1994, Plant Journal 6:271-282).
[0025] The obtained transgenic rice plant was designated as T050U.
The present invention obtained in total 23 independent transgenic
rice plants.
Example 4
Cold Resistance Screening of the SNAC2 Gene Transgenic
T2 Family in Seedling Stage
[0026] In order to verify whether the cold resistance of transgenic
rice plants was enhanced and whether such a enhancement was related
to an introduced SNAC2 gene, the expression of SNAC2 gene in a
portion of transgenic rice plants was detected by Northern
hybridization technology in the present invention (FIG. 3 shows the
Northern hybridization results, wherein the method was the same as
used in Example 2), and a portion of families of T2 generation
plants of the present invention was screened for the cold
resistance. The specific steps were as follows: the seeds of T2
generation families were germinated in MS medium containing 50
mg/ml hygromycin for 5 days, after which seedlings having
substantially the same level of germination were transplanted into
little red pails, one half of which was planted with transgenic
over-expressed plants, and the another half was planted with
wild-type control plants. When the plants grew to 4-leaf stage,
they were subjected to low temperature treatment at 4.degree. C.
After 5 days of treatment, it was observed that no significant
changes occurred to the phenotypes of either the transgenic plants
or to the control plants. However, after they were shifted to
normal conditions to recover growth for 3 days, it was observed
that most of the control plants had leaves that were curled and
became wilted, while only a few of leaves of the transgenic plants
got wilted; at 7 days after the growth recovery, almost all of the
control plants died, while, in contrast, almost 50% of the
transgenic over-expressed family survived (FIG. 4). This proved
that SNAC2 gene was without doubt related to cold resistance, and
its over-expression could enhance the cold resistance of transgenic
plants, and that the enhancement of resistance of transgenic rice
plants was surely related to the introduced SNAC2 gene.
Example 5
Salinity Resistance Screening of SNAC2 Gene Transgenic T2 Family in
Seedling Stage
[0027] It was proved in Example 4 that the cold resistant
performance of SNAC2 gene transgenic plants of the present
invention in seedling stage was significantly higher than that of
the control. In order to verify whether SNAC2 is capable of
protecting plants against other stresses, growth performance of
plants in high-salinity environment was compared in this example.
The comparison is taught as follows: T2 generation of transgenic
over-expression family was germinated in MS medium containing 50
mg/l hygromycin for 4 days, then the transgenic seedlings and the
control seedlings, which had the same growth, were transplanted to
little square boxes having MS medium containing 150 mmol/L NaCl to
keep growing. Growth was observed, and 18 days later, a measure was
made to the root length and plant height of each young seedling. It
was found that there was no difference in root length between the
SNAC2 over-expression or SNAC2 inducible expression transgenic
plant and the control plant grown in the high salinity condition.
However, there was an apparent difference in the plant height. The
growth of the control plant was substantially inhibited in the high
salinity condition, reaching only 60% of that of the transgenic
over-expression family (FIG. 5). This showed that the SNAC2
over-expression transgenic seedling had higher salinity tolerance
than the control plant, suggesting that the SNAC2 transgenic plant
of the present invention can substantially improve salinity
resistance in the plants.
Example 6
SNAC2 Gene Having Transcription Activating and DNA Binding
Properties
[0028] Transcription factors have transcription activating and DNA
binding properties. Specifically, transcription factors bind cis
acting elements of downstream gene promoters in case of signaling
or stress inducement, and thereby initiate the expression of
downstream target genes. Since the gene of the present invention is
an inducible transcription factor, in order to verify whether the
SNAC2 gene of the present invention has transcription activating
and DNA binding properties, a trans-activation assay and a yeast
one hybrid assay were conducted in the present example to verify
the DNA binding activity and transcription regulating (activating)
function of a SNAC2 protein as a transcription factor. At first,
the SNAC2 gene was constructed into a yeast GAL4-DB fusion
expression vector pDEST32 (purchased from Invitrogen Inc.), which
was used to transform a yeast cell Y187 (purchased from CLONTECH
Inc.). Then, a .beta.-Galactosidase activity assay was conducted to
determine the expression of reporter gene LacZ based on whether the
yeast colony turned blue, thereby determining whether the gene had
activation function. The assay results showed that the gene of the
present application does activate transcription (FIG. 6A). The
research results of Hu et al. ("Overexpressing a NAM, ATAF, and CUC
(NAC) transcription factor enhances drought resistance and salt
tolerance in rice." Proc Natl Acad Sci USA, 2006, 103: 12987-12992)
suggest that SNAC1 could bind to a DNA sequence similar to a NACRS
(NAC recognition site) identified in Arabidopsis. In order to
further verify whether the other NAC type protein SNAC2 in rice
could also bind to this sequence, the applicants further examined
in the present example the interaction between the SNAC2 protein
and the DNA containing sequence of CATGTG and CACG in OsERD1
promoter in yeast. The yeast cell Y187 was co-transformed with
pHIS-cis and expression vector pGAD-SNAC2 in which the full-length
SNAC2 encoding sequence was fused to GAL4-activation domain of
yeast vector pGAD-RecT7 by the present applicants. At the same time
the positive control (pHIS53/p53GAD) and negative control
(pGAD-SNAC2/pHIS53) were transformed. The result showed that the
desired transformer, negative control and positive control can grow
in petri dishes SD/Leu.sup.-/Trp.sup.-/His.sup.- for yeast without
3-AT. However, when 20 mmol/L 3-AT was added, the negative control
transformer could not grow in SD/Leu.sup.-/Trp.sup.-/His.sup.-
medium, while the positive control and the desired transformer can
grow well (FIG. 6B). This result showed that SNAC2 can also
recognize and bind to the sequence of OsERD1 promoter region
containing CATGTG and CACG, and had transcription activating
function in yeast cell. The result also suggested that the NAC
protein of rice can recognize sequences similar with that
recognized by Arabidopsis NAC protein. All of these experiments
showed that the SNAC2 gene has transcription activating and DNA
binding properties.
[0029] The specific steps for executing the trans-activation assay
were as follows:
[0030] 1. The full-length SNAC2 gene was fused to the yeast
expression vector pDEST32 (purchased from Invitrogen Inc.).
[0031] The gene primers were designed according to the open reading
frame of pDEST32 vector based on the full-length cDNA clone
sequence (using software Primer 5.0). The obtained PCR products
were purified by PEG8000, before subjected to a BP recombination
reaction with an intermediate vector pDONR221 (purchased from
Invitrogen Inc.). The reaction system was in a volume of 5 .mu.l,
including 200 ng of PCR product, 50 ng of pDONR221, 2 .mu.l of
5.times.BP Clonase Reaction Buffer and 2 .mu.l of BP Clonase Mix,
and was incubated for reaction at 25.degree. C. for about 5 h. An
E. coli DH10.beta. (purchased from Invitrogen Inc.) was transformed
with reaction product to screen out the positive clones. The
desired positive clone plasmids were finally subjected to a LR
recombination reaction, such that the gene fragment carried by the
desired positive clone plasmid was fused to yeast expression vector
pDEST32. Said LR recombination reaction is taught as follows: an E.
coli DH10.beta. (purchased from Invitrogen Inc.) was transformed at
25.degree. C. for about 5 h with 100 ng of positive plasmid of BP
reaction, 50 ng of pDEST32, 2 .mu.l of 5.times.LR Clonase Buffer
and 2 .mu.l of LR Clonase Mix, and the positive clones were
screened.
[0032] 2. Preparation and transformation of competent yeast
(CLONTECH, Yeast Protocols Handbook) by lithium acetate (LiAc)
method
[0033] 1) Reagent and Formula
[0034] A. YPD medium:
TABLE-US-00001 20 g Difco peptone 10 g Yeast extract 20 g glucose
diluted with distilled water to 1 L, and sterilized for 15
minutes.
[0035] B. SD/Leu medium:
TABLE-US-00002 6.7 g Yeast nitrogen base without amino acids 20 g
Agar powder 20 g glucose 0.69 g -Leu DO Supplement (purchased from
CLONTECH Inc.) diluted with distilled water to 1 L, and sterilized
for 15 minutes.
[0036] C. 10 TE buffer: 0.1 M Tris-HCl, 10 mM EDTA, pH 7.5,
sterilized
[0037] D. 10 LiAc: 1M lithium acetate, pH 7.5, sterilized
[0038] E. PEG/LiAc solution
TABLE-US-00003 Final Conc. To prepare 10 ml of solution PEG4000 40%
8 ml of 50% PEG TE buffer 1X 1 ml of 10X TE LiAc 1X 1 ml of 10X
LiAc
[0039] 2) Steps:
[0040] A. Single yeast colony with diameter of 2-3 mm was scattered
with 1 ml YPD solution, and then transferred to a triangular flask
containing 10 ml YPD medium.
[0041] B. Cultured under the rotation of 250 rpm at 30.degree. C.
for 16-18 h, such that OD600>1.5.
[0042] C. About 5 ml of the above-mentioned yeast solution was
transferred to another triangular flask containing 50 ml YPD
medium, and detected concentration to get OD600=0.2-0.3.
[0043] D. Cultured at 30.degree. C. for 3 h (230 rpm), at this
point OD600=0.4-0.6 (if OD600<0.4, the culture maybe get in
trouble).
[0044] E. The yeast solution was transferred into a 50 ml
centrifuge tube, and centrifuged at 1000.times.g for 5 minutes at
room temperature.
[0045] F. The supernatant was discarded, the cells were resuspended
with sterilized double distilled water, and centrifuged at
1000.times.g for 5 minutes at room temperature.
[0046] G. The supernatant was discarded, the yeast cells were mixed
homogenously with 1 ml fresh prepared 1.times.TE/1.times.LiAc.
[0047] H. 200 ng fusion plasmid DNA was placed into a 1.5 ml
centrifuge tube, and 100 .mu.l of yeast competent cells were added
and mixed homogeneously, then 600 .mu.l PEG/LiAc was added, mixed
homogeneously by centrifugation at high speed, and cultured at
30.degree. C. for 30 min (200 rpm).
[0048] I. 70 .mu.l DMSO (100%) was added, mildly reversed for
several times, placed in 42.degree. C. water bath for 15 min, and
then placed on ice for 2 min.
[0049] J. Centrifuged at 14000 rpm for 5 seconds at room
temperature, the supernatant was discarded, and the cells were
scattered with 500 .mu.l 1.times.TE buffer.
[0050] K. 100 .mu.l transformed cells were uniformly coated on
-Leu/SD plate, inversion cultured in 30.degree. C. incubator for
2-4 days, until clones appeared.
[0051] 3. Verification of transcription activity of SNAC2 gene and
deletion mutant thereof based on the expression of reporter gene
LacZ in beta-galactosidase assay
[0052] 1) Reagent and Formulation
[0053] A. Z buffer
TABLE-US-00004 Na.sub.2HPO.sub.4.cndot.7H.sub.2O 16.1 g/L
NaH.sub.2PO.sub.4.cndot.H.sub.2O 5.5 g/L KCl 0.75 g/L
MgSO.sub.4.cndot.7H.sub.2O 0.246 g/L adjusted pH to 7.0, and
sterilized.
[0054] B. X-gal stock solution (20 mg/ml)
[0055] C. Z buffer solution/X-gal solution:
TABLE-US-00005 100 ml Z buffer: 0.27 ml .beta.-mercaptoethanol 1.67
ml X-gal stock solution
[0056] 2) Steps:
[0057] A. The transformed clone was allowed to grow to 1-3 mm
(30.degree. C., 2-4 days)
[0058] B. Round sterilized Watman filter paper of appropriate size
was placed on 10 cm asepsis plate, about 2.5-5 ml Z buffer/X-gal
solution was added to wet the filter paper, and bubble was
avoided.
[0059] C. Another clean, sterilized filter paper was placed by
forceps on the petri dish with growing clone, and the filter paper
was slightly pressed in order to adhere the clone to the filter
paper.
[0060] D. When the filter paper was wetted, it was uncovered by
forceps, and the filter paper was placed into liquid nitrogen for
10 sec with the surface adhered with the clones facing up, then it
was thawed at room temperature in order to break the yeast
cells.
[0061] F. The filter paper with surface adhered with clones facing
up was carefully placed onto the previously wetted filter paper,
and bubble was avoided.
[0062] G. The filter paper was placed at 30.degree. C. (30 min-8
hr), and whether the gene had the activation function was
determined according to the occurrence of blue spot.
[0063] The specific implementing steps of yeast one hybrid assay
were as follows:
[0064] 1. The full length SNAC2 gene was fused to the yeast
expression vector pGAD-Rec2 (purchased from CLONTECH Inc.).
[0065] The gene primers (5-TAGAATTCGACGAGGAGCTGGTGATGC-3, specific
primer plus EcoRI enzymatically cleavage site) and
(5-TAGGATCCCCTCGTCGTCGTTCAGTCC-3, specific primer plus BamHI
enzymatically cleavage site) were designed according to the open
reading frame of pGAD-Rec2 vector based on the full length cDNA
clone sequence. The obtained PCR products were double digested,
purified by chloroform: isopentanol and linked to the vector
pGAD-Rec2 subjected to same double digestion. E. coli DH10.beta.
(purchased from Invitrogen Inc.) was transformed, and the positive
clones were screened and verified with same enzyme digestion
method, yielding the yeast transformation vector pGAD-SNAC2.
[0066] 2. The 3 repeats of 90 by OsERD1 promoter region sequence
containing the core sequence CATGTG and CACG was linked into vector
pHIS2 (purchased from CLONTECH Inc.).
[0067] The 3 tandem repeats of 90 by OsERD1 promoter region
(5'-CCCCGCGCGACGTCGACAAGTCGACAAGTGCGAGGAGCTAG
CCATGTGGGTCGTGCCCGCGCGCGCCACGGCACGGCAACCCCG GAAACG-3') comprising
the core sequence CATGTG and CACG was synthesized inhouse with
EcoRI and Sad site at both end, and it was directedly linked into
yeast vector pHIS2. The positive clones were verified and screened
with same enzyme digestion method, yielding the yeast transform
vector pHIS2-cis.
[0068] 3. The competent cells were prepared and the transformation
was accomplished with a yeast vector (the method was same as
trans-activation assay). The cells transformed with desired binary
vector (at the same time preparing positive control and negative
control) were coated on petri dish of SD/Leu-/Trp-, then cultured
in incubator at 30.degree. C., until size of colony was about 2
mm.
[0069] 4. The same colony was streak cultured in a petri dish of
SD/Leu-/Trp-/His-containing 0 mM, 10 mM, 20 mM, 30 mM and 40 mM
3-AT, and growth performance of colony was observed.
Example 7
Subcellular Localization of SNAC2
[0070] In order to determine the expression location of the SNAC2
gene in a cell, a GFP-NLS (nuclear location signal) fusion protein
was constructed. That is, the gene expression profile in cell was
determined according to the expression of GFP. It was known that
the nuclear location signal (NLS) of the NAC gene may locate at
71-83 AA according to the previously published articles on NAC gene
(Mild Fujita, Kazuo Shinozaki et al., A Dehydration-induced NAC
protein, RD26, is involved in a novel ABA-dependent
stress-signaling pathway." Plant J (2004) 39, 863-876, and Honghong
Hu et al., "Over expressing a NAM, ATAF, and CUC (NAC)
transcription factor enhances drought resistance and salt tolerance
in rice." Proc Natl Acad Sci USA, 2006, 103: 12987-12992). In this
context, the subcellular location of the gene was determined
according to the expression region in cell of this sequence fused
with GFP. The 1-144 AA fragment of the sequence of the present
invention was fused to a pCAMBIA1391-GFP vector (bearing ubiquitin1
promoter), whereby the cellular location of SNAC2 protein can be
deduced according to the expression location of GFP in cell in
P.sub.SNAC2:.DELTA. SNAC2-GFP transgenic plants. The
pCAMBIA1391-EGFP vector (see figure below) was reconstructed based
on the pCAMBIA1391 (a plant genetic transformation vector commonly
used in the world), wherein the carried GUS gene was replaced with
EGFP gene, with Ubiquitin1 promoter preceding GFP. The pCAMBIA1391
vector was from Australia CAMBIA Laboratory (Center for the
Application of Molecular Biology to International Agriculture) and
is public available.
##STR00002##
[0071] The method for the construction of the fused gene vector was
described as follows: the primers PF (5-GGATCCCTCCTCTCCTCTTCCCAT,
plus linker BamHI site) and PR (5-GAATTCGTTCTTCTTGCGG, plus linker
EcoRI) were designed; the vector pGEM-SNAC2 constructed in above
Example 1 was used as template. The SNAC2 gene was amplified by
means of an amplification program of predenaturation at 94.degree.
C. for 3 min; 30 cycles of 94.degree. C. for 30 sec, 55.degree. C.
for 30 sec, 72.degree. C. for 3 min; extension at 72.degree. C. for
5 min; and the amplified product was double digested by EcoRI and
HindIII and linked into a pCAMBIA1391-EGFP vector that had been
subjected to the same double digestion. .degree. The rice callus
was transformed with the fusion vector p1391-GFP-NLS using an
agrobacterium mediated genetic transformation method (same as the
method used in Example 3), the callus with resistance was obtained
under hygromycin selection pressure (specific methods as described
in Example 3), and the expression of GFP was observed under
fluorescence microscope (see FIG. 7A). The expressed resistance
callus was sectioned and observed under confocal microscope to
determine the intracellular expression of GFP. FIG. 7B shows that
GFP is expressed only in nuclei under the observation of confocal
microscope, which indicates that the sequence of 1-144AA already
includes the NLS, so GFP could be localized in the nuclei, i.e.,
the SNAC2 protein was localized in nuclei. This example proved that
the 1-144AA fragment of the sequence according to the present
invention includes an intact NLS, and the SNAC2 protein localizes
in cell nucleus.
Sequence CWU 1
1
911529DNAOryza sativaCDS(112)..(1023) 1gccaagccct cctctcctct
tcccaacact agtaggataa agccacagag agagcagtag 60tagtagcgag ctcgccggag
aacggacgat caccggagaa gggggagaga g atg agc 117 Met Ser 1ggc ggt cag
gac ctg cag ctg ccg ccg ggg ttc cgg ttc cac ccg acg 165Gly Gly Gln
Asp Leu Gln Leu Pro Pro Gly Phe Arg Phe His Pro Thr 5 10 15gac gag
gag ctg gtg atg cac tac ctc tgc cgc cgc tgc gcc ggc ctc 213Asp Glu
Glu Leu Val Met His Tyr Leu Cys Arg Arg Cys Ala Gly Leu 20 25 30ccc
atc gcc gtc ccc atc atc gcc gag atc gac ctc tac aag ttc gat 261Pro
Ile Ala Val Pro Ile Ile Ala Glu Ile Asp Leu Tyr Lys Phe Asp35 40 45
50cca tgg cag ctt ccc cgg atg gcg ctg tac gga gag aag gag tgg tac
309Pro Trp Gln Leu Pro Arg Met Ala Leu Tyr Gly Glu Lys Glu Trp Tyr
55 60 65ttc ttc tcc ccg cga gac cgc aag tac ccg aac ggg tcg cgg ccg
aac 357Phe Phe Ser Pro Arg Asp Arg Lys Tyr Pro Asn Gly Ser Arg Pro
Asn 70 75 80cgc gcc gcc ggg tcg ggg tac tgg aag gcg acc ggc gcc gac
aag ccg 405Arg Ala Ala Gly Ser Gly Tyr Trp Lys Ala Thr Gly Ala Asp
Lys Pro 85 90 95gtg ggc tcg ccg aag ccg gtg gcg atc aag aag gcc ctc
gtc ttc tac 453Val Gly Ser Pro Lys Pro Val Ala Ile Lys Lys Ala Leu
Val Phe Tyr 100 105 110gcc ggc aag gcg ccc aag ggc gag aag acc aac
tgg atc atg cac gag 501Ala Gly Lys Ala Pro Lys Gly Glu Lys Thr Asn
Trp Ile Met His Glu115 120 125 130tac cgc ctc gcc gac gtc gac cgc
tcc gcc cgc aag aag aac agc ctc 549Tyr Arg Leu Ala Asp Val Asp Arg
Ser Ala Arg Lys Lys Asn Ser Leu 135 140 145agg ttg gat gat tgg gtg
ctg tgc cgg att tac aac aag aag ggc ggg 597Arg Leu Asp Asp Trp Val
Leu Cys Arg Ile Tyr Asn Lys Lys Gly Gly 150 155 160ctg gag aag ccg
ccg gcc gcg gcg gtg gcg gcg gcg ggg atg gtg agc 645Leu Glu Lys Pro
Pro Ala Ala Ala Val Ala Ala Ala Gly Met Val Ser 165 170 175agc ggc
ggc ggc gtc cag agg aag ccg atg gtg ggg gtg aac gcg gcg 693Ser Gly
Gly Gly Val Gln Arg Lys Pro Met Val Gly Val Asn Ala Ala 180 185
190gtg agc tcc ccg ccg gag cag aag ccg gtg gtg gcg ggg ccg gcg ttc
741Val Ser Ser Pro Pro Glu Gln Lys Pro Val Val Ala Gly Pro Ala
Phe195 200 205 210ccg gac ctg gcg gcg tac tac gac cgg ccg tcg gac
tcg atg ccg cgg 789Pro Asp Leu Ala Ala Tyr Tyr Asp Arg Pro Ser Asp
Ser Met Pro Arg 215 220 225ctg cac gcc gac tcg agc tgc tcg gag cag
gtg ctg tcg ccg gag ttc 837Leu His Ala Asp Ser Ser Cys Ser Glu Gln
Val Leu Ser Pro Glu Phe 230 235 240gcg tgc gag gtg cag agc cag ccc
aag atc agc gag tgg gag cgc acc 885Ala Cys Glu Val Gln Ser Gln Pro
Lys Ile Ser Glu Trp Glu Arg Thr 245 250 255ttc gcc acc gtc ggg ccc
atc aac ccc gcc gcc tcc atc ctc gac ccc 933Phe Ala Thr Val Gly Pro
Ile Asn Pro Ala Ala Ser Ile Leu Asp Pro 260 265 270gcc ggc tcc ggc
ggc ctc ggc ggc ctc ggc ggc ggc ggc agc gac ccc 981Ala Gly Ser Gly
Gly Leu Gly Gly Leu Gly Gly Gly Gly Ser Asp Pro275 280 285 290ctc
ctc cag gac atc ctc atg tac tgg ggc aag cca ttc tag 1023Leu Leu Gln
Asp Ile Leu Met Tyr Trp Gly Lys Pro Phe 295 300acgaccaaaa
aaaaaaaaaa acaaccgcat tggcagcaat ggtgtcactg aacaccgtgc
1083aggctagcta gcttcatggc cggtgaactt tgactcaggc gagccgccgg
agttgactca 1143aagataatta aaagaagtgt tttaagtgga ttggattgga
ttagacagag gagatgagga 1203ctcgagaaag gcggcgatga gaccgtggtt
ggggggaccc tggcctggac tgaacgacga 1263cgaggcagca gcagaaagat
ggtgcaattg catcgggtgg catgtcagtg tgtgtgtata 1323gtggcatgta
catagtacat ggtgattgat tcggtataca gggggctagc tttcctgttt
1383ctgtttcttc attggttaat tattactccc attataaggt cttcttcagg
gttgctagct 1443taattaatta attaattagc ccagtggttg aagtgtaagt
caaaattcat caagtcagag 1503actggaataa tacaatacag tactgc
15292303PRTOryza sativa 2Met Ser Gly Gly Gln Asp Leu Gln Leu Pro
Pro Gly Phe Arg Phe His1 5 10 15Pro Thr Asp Glu Glu Leu Val Met His
Tyr Leu Cys Arg Arg Cys Ala 20 25 30Gly Leu Pro Ile Ala Val Pro Ile
Ile Ala Glu Ile Asp Leu Tyr Lys 35 40 45Phe Asp Pro Trp Gln Leu Pro
Arg Met Ala Leu Tyr Gly Glu Lys Glu 50 55 60Trp Tyr Phe Phe Ser Pro
Arg Asp Arg Lys Tyr Pro Asn Gly Ser Arg65 70 75 80Pro Asn Arg Ala
Ala Gly Ser Gly Tyr Trp Lys Ala Thr Gly Ala Asp 85 90 95Lys Pro Val
Gly Ser Pro Lys Pro Val Ala Ile Lys Lys Ala Leu Val 100 105 110Phe
Tyr Ala Gly Lys Ala Pro Lys Gly Glu Lys Thr Asn Trp Ile Met 115 120
125His Glu Tyr Arg Leu Ala Asp Val Asp Arg Ser Ala Arg Lys Lys Asn
130 135 140Ser Leu Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn
Lys Lys145 150 155 160Gly Gly Leu Glu Lys Pro Pro Ala Ala Ala Val
Ala Ala Ala Gly Met 165 170 175Val Ser Ser Gly Gly Gly Val Gln Arg
Lys Pro Met Val Gly Val Asn 180 185 190Ala Ala Val Ser Ser Pro Pro
Glu Gln Lys Pro Val Val Ala Gly Pro 195 200 205Ala Phe Pro Asp Leu
Ala Ala Tyr Tyr Asp Arg Pro Ser Asp Ser Met 210 215 220Pro Arg Leu
His Ala Asp Ser Ser Cys Ser Glu Gln Val Leu Ser Pro225 230 235
240Glu Phe Ala Cys Glu Val Gln Ser Gln Pro Lys Ile Ser Glu Trp Glu
245 250 255Arg Thr Phe Ala Thr Val Gly Pro Ile Asn Pro Ala Ala Ser
Ile Leu 260 265 270Asp Pro Ala Gly Ser Gly Gly Leu Gly Gly Leu Gly
Gly Gly Gly Ser 275 280 285Asp Pro Leu Leu Gln Asp Ile Leu Met Tyr
Trp Gly Lys Pro Phe 290 295 300334DNAArtificialsynthetic
3caggtaccgc caagccctcc tctcctcttc ccat 34427DNAArtificialsynthetic
4caggatcccc tcgtcgtcgt tcagtcc 27527DNAArtificialsynthetic
5tagaattcga cgaggagctg gtgatgc 27627DNAArtificialsynthetic
6taggatcccc tcgtcgtcgt tcagtcc 27790DNAArtificialsynthetic
7ccccgcgcga cgtcgacaag tcgacaagtg cgaggagcta gccatgtggg tcgtgcccgc
60gcgcgccacg gcacggcaac cccggaaacg 90824DNAArtificialsynthetic
8ggatccctcc tctcctcttc ccat 24919DNAArtificialsynthetic 9gaattcgttc
ttcttgcgg 19
* * * * *
References